Metabolic profiling of Lantana camara L. using UPLC-MS/MS and revealing its inflammation-related targets using network pharmacology-based and molecular docking analyses

Lantana camara L. is widely used in folk medicine for alleviation of inflammatory disorders, but studies that proved this folk use and that revealed the molecular mechanism of action in inflammation mitigation are not enough. Therefore, this study aimed to identify L. camara phytoconstituents using UPLC-MS/MS and explain their multi-level mechanism of action in inflammation alleviation using network pharmacology analysis together with molecular docking and in vitro testing. Fifty-seven phytoconstituents were identified in L. camara extract, from which the top hit compounds related to inflammation were ferulic acid, catechin gallate, myricetin and iso-ferulic acid. Whereas the most enriched inflammation related genes were PRKCA, RELA, IL2, MAPK 14 and FOS. Furthermore, the most enriched inflammation-related pathways were PI3K-Akt and MAPK signaling pathways. Molecular docking revealed that catechin gallate possessed the lowest binding energy against PRKCA, RELA and IL2, while myricetin had the most stabilized interaction against MAPK14 and FOS. In vitro cytotoxicity and anti-inflammatory testing indicated that L. camara extract is safer than piroxicam and has a strong anti-inflammatory activity comparable to it. This study is a first step in proving the folk uses of L. camara in palliating inflammatory ailments and institutes the groundwork for future clinical studies.


In vitro cytotoxicity and anti-inflammatory activity testing
It was carried out according to the method described by Darwish et al. 45

as following:
Isolation and cultivation of human white blood cells A blood specimen was provided from Alexandria Regional Blood Transfusion Center (63 Ahmed Soliman El-Shaikh Street, Kom Ad Dakah Sharq, Al Attarin, Alexandria Governorate, Egypt).
This blood specimen was placed in a sterile heparin tube, from which 1 mL was drawn into 15 mL centrifuge tube which was then filled with 10%v/v fresh cold lysing solution prepared from stock solution containing NH4Cl 8.02g, NaHCO3 0.84g and EDTA 0.37g. The centrifuge tube was then inverted at room temperature for about 10 min till the liquid turned into clear red. After that, the blood specimen was centrifuged at 2000 rpm and 4 o C for 10 min and the supernatant was decanted.
Then they were resuspended in RPMI-1640 medium, containing 10% fetal bovine and 2% Lglutamine. The evaluation of WBCs viability and counting was carried out using dye exclusion method 46 . Fifty µL of cell suspension was blended with equal volume of 0.5% trypan blue staining solution then loaded onto hemocytometer. Eventually, counting of viable "unstained" and nonviable "stained" cells in each of the four corner quadrants (A, B, C, D) was carried out.

% Cell viability = (Number of viable cells / Total number of cells) × 100
% Cell viability must be at least 90% in order to perform the assays. WBCs were cultured in RPMI media and incubated in CO2 incubator at 37°C, 5% CO2, and 90% relative humidity for six days.
Then they were seeded in 96 well cell culture plate (100,000 cells/ well).

Assessment of cytotoxicity of the crude extracts compared to piroxicam (MTT assay)
Treatment of 200 µL cultured medium containing 100,000 WBCs / well with different RPMI medium control. Plates were then incubated in CO2 incubator for 72 of MTT solution was added to each well and re-incubated to allow MTT reaction to be accomplished. Afterwards, the plates were centrifuged at 1650 rpm for 10 min and the medium measurement of absorbance was performed at a wavelength of 570 nm using optima spectrophotometer, in order to detect the safe dose which causes 100% cell viability.
The % viability was calculated as follow: (AT-Ab /AC-Ab) x 100 AT = mean absorbances of cells treated with a certain concentration of the plant extract.

AC = mean absorbances of control untreated cells with culture medium only
Ab= mean absorbances of cells treated with vehicle of plant extract (RPMI without fetal bovine serum) The cytotoxicity assay of the compound was expressed as CC50, which is the drug concentration required for reducing the cell viability by 50%, and it was calculated by the Graphpad Instat software (https://www.graphpad.com/scientific-software/instat/) by interpolation from the plot of % cell viability vs serial dilutions of the plant extract. Cell pellets were suspended in 50 µL of solution R1 (qiagen RNA extraction kit). They were mixed for 30 s, then incubated for 1 min at room temperature. Afterwards, 300 µL of solution R2 (qiagen RNA extraction kit) were added and blended for 30 s. Centrifugation was accomplished for 3-5 min at 4ºC. Next, the supernatant was transferred into a spin column and centrifuged at 14000 rpm for 30 s at 4ºC. Then, addition of 300 µL of working wash buffer was done after removing the flow-through and centrifugation was repeated for 30 s. This step was repeated twice. Subsequently, the spin column was centrifuged at 10,000 rpm for 1 min then delivered to a sterile 1.5 mL micro centrifuge tube. Then, 30 µL of elution buffer were added to the membrane center and incubated for 1 min at room temperature, then centrifuged at 14000 rpm for 30 s at 4ºC. Eventually, the optical density (OD) of the extracted RNA was determined via absorbance and purity measurement at A260 and A260/A280 nm, respectively. Then it was kept at -80°C until real time polymerase chain reaction (PCR) was performed.
cDNA synthesis from RNA extracted from untreated and treated LPS-stimulated human white blood cells In PCR tubes, 2 µg of total RNA or nuclease-free water and 1 µL of oligo dT primer were added to nuclease-free water in a total volume of 12 µL, and they were gently mixed. Centrifugation was performed before incubation for 5 min at 65ºC in PCR machine, then the mixture was set immediately on ice. Four microliters of 5X reaction buffer, 1 µL of RNase inhibitor, 2 µL of dNTPs mix and 1 µL of reverse transcriptase or 1 µL of nuclease-free water instead of reverse transcriptase for reverse transcriptase negative control were gently mixed with previous mixture. Afterwards, the PCR tubes were spined down and incubated for 60 min at 42ºC, then they were heat inactivated at 70ºC for 5 min in PCR machine Determination of IL-1 , IL 6, TNF and INF-expression level by real time polymerase chain reaction (PCR) In PCR tubes, admixture of 13 µL of 2 X SYBR green master mix with 5 µL of cDNA, 0.5 µL of 10 pmoles/mL forward primer and 0.5 µL of 10 pmoles/mL reverse primer for each primer was carried out. The same as in the reference tube, addition of 0.5 µL of 10 pmoles/mL forward primer --actin was done. Another tube was utilized as a non-template control (NTC) to assess reagent contamination or primer dimers, by inserting 1 µL of nuclease-free water as a substitute of template used. Afterwards, the tubes were subjected to gentle mixing with 6.5 µL nuclease free water without bubbles formation and subsequently subjected to spinning for few seconds. Samples were set in the cycler and the program was initiated as following; initial denaturation (1 cycle of 95ºC for 10 min), then denaturation (40 cycles of 95ºC for 15 sec), annealing (at 60ºC for 30s) and extension (at 72ºC for 30s). Fold change in gene expression was used to assess the influence of LPS and extracts on the expression of genes.

Calculation
Expressions fold levels of gene are computed by normal =Ct normal untreated cells Ct reference tested plant extract = Ct tested plant extract-treated cells Ct reference induced =-Ct LPS-exposed cells Ct reference In case of genes